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An article to be submitted to Clinical and Vaccine Immunology

Liying Lei 1,2, Brandon L. Plattner 2, and Jesse M. Hostetter 1,2

Abstract

In this study, we evaluated the phenotypic and functional features of the CD11c+ dendritic cell (DC)-like cells, cellular composition and the cytokine environment in the lymph node draining Mycobacterium avium subspecies paratuberculosis (M.

paratuberculosis) challenge site. One group of calves was made immune by prior vaccination

with M. paratuberculosis vaccine followed by M. paratuberculosis challenge. The control

group was non-vaccinated calves challenged with M. paratuberculosis. Granulomatous

lesions persisted at the challenge sites in both vaccinated and non-vaccinated animals from day 7 till day 60 post inoculation, the conclusion of the study. In vaccinated animals, the cervical lymph node draining the challenge site (CLN) appeared to be in an activated state with more germinal centers than the CLN in non-vaccinated animals. By flow cytometric analysis, greater numbers of CD11c+ CD205+ cells and B cells were present in the vaccinated animal CLN. Similar numbers of macrophages, CD4 T cells, CD4+ CD25+ T cells, and CD8 T cells were present in both vaccinated and non-vaccinated animal CLNs. The CD11c+ cells had a mature phenotype with high levels of MHC I, MHC II, and CD205 surface expression.

1_{Immunobiology Graduate Program, Iowa State University, Ames, Iowa}

2_{Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State} University, Ames, Iowa

Vaccinated animals CLN CD4+ T lymphocytes proliferated at a significantly higher level after stimulation with homogeneous CLN PPD-pulsed CD11c+ cells compared to non- vaccinated animal lymphocytes. Also, these lymphocytes that underwent proliferation produced high levels of IFN-γ and IL-4. Interestingly, non-vaccinated animals CLN CD8+ T lymphocytes proliferated at a higher level upon PPD-pulsed CD11c+ cells stimulation. Cells in the lymph node draining M. paratuberculosis challenge site produced high levels of

cytokine IL-12 and IL-10, and which was independent of the prior vaccination status. These results suggest that commercial M. paratuberculosis vaccination has effects on DC activation

and maturation upon M. paratuberculosis challenge. CD4 T cell and CD8 T cell recall

response and cytokine environment in the lymph node draining M. paratuberculosis

challenge site are affected by prior vaccination.

Key words: Mycobacterium paratuberculosis, dendritic cell, CD11c+ cell, innate immunity,

granuloma, vaccine.

Introduction

Early M. paratuberculosis infection is associated with dominant cell mediated T-

helper cell type 1 (Th1) immune response, which is the response essential to control infections by intracellular pathogen. With the progression of the infection, Th1 immune response wanes and overlaps with a humoral (Th2) immune response (Jiao et al., 2003; Ritacco et al., 1991). Development of clinical disease is generally consider to be associated with a dominant Th2 immune response (Burrells et al., 1998; Chiodini, 1996). The nature of early immune response to M. paratuberculosis and the exact mechanisms responsible for the

shift of balance between Th1 and Th2 response are still unclear. In previous studies we found that live M. paratuberculosis infection induced limited activation and maturation of dendritic

cell in both in vitroand in vivo infection systems (Lei and Hostetter, 2007 and data in

preparation for publication). In this study our goal is to analyze the phenotype and function of the CD11c+ DC-like cells in the lymph node draining M. paratuberculosis infection site.

Since semi-mature DC can also migrate into draining lymph node and induce the

differentiation of T cells with a regulatory phenotype, it is of great interest to determine the maturation status of these lymph node DC and the type of T cell immune response these DC induce.

With the implementation of the Johne’s disease control programs and the lack of treatment options, there is renewed interest in prevention of M. paratuberculosis infection.

Novel vaccine designs are under development including subunit vaccine and DNA vaccine strategies. A more complete understanding of the immune response following vaccination will provide valuable information in future vaccine design. We were interested in

understanding the mechanism of the vaccine in shaping the host immune response, specifically the phenotype and function of antigen presenting cells. Mycobacterium bovis

Bacillus Calmette-Guerin (BCG) vaccination was shown to promote development of IL-10- producing dendritic cells and IL-10-producing T cells, while inhibiting IL-12 production by these monocyte-derived DC (Madura Larsen et al., 2007). BCG vaccination also enhanced macrophage activity inside the granulomatous lesions upon Mycobacterium tuberculosis (M. tuberculosis) challenge (Ordway et al., 2006). In this study, BCG vaccinated mice had a

better control of following M. tuberculosis infection with increasing macrophage number and

lower bacterial load. The goal of the current study was to determine the effects of M.

paratuberculosis vaccine on the phenotype, function, activation, and maturation of dendritic

cells upon M. paratuberculosis challenge.

To achieve this goal, we used the localized M. paratuberculosis subcutaneous

infection model system developed by Simutis et al. with modifications (Simutis et al., 2005). Previous research using this model system demonstrated that focal granulomatous lesions developed in response to subcutaneous bacterial challenge and persisted from day 7 untill day 60 post inoculation, the conclusion of the study. Using this system, dendritic cells and lymphocytes in the lymph node draining M. paratuberculosis inoculation site can be readily

obtained for phenotypic and functional assays. CD11c+ cells were isolated by magnetic cell isolation and used for functional and phenotypic analyses. We determined the cellular

composition of the lymph node draining M. paratuberculosis inoculation site, surface marker

expression on isolated CD11c+ cells, the ability of these cells to induce antigen-specific CD4 and CD8 T cell proliferation and cytokine production.

The results of these studies indicate that significantly greater numbers of CD11c+ cells were present inside the vaccinated calves lymph node draining M. paratuberculosis

inoculation site (CLN) than the CLN in non-vaccinated calves. These cells had a mature phenotype with high level expression of MHC I, MHC II, and CD205, and high ability to induce antigen-specific CD4 T cell proliferation and IFN-γ and IL-4 production. CD8 T cells in vaccinated animal CLN proliferated at a lower level upon PPD-pulsed DC stimulation than non-vaccinated animal.

Materials and Methods

Animals. 4-6 weeks old Holstein calves were used in the following experiments and were housed in isolation in Iowa State University College of Veterinary Medicine biosafety level II animal care facility during the study. A total of nine calves were used for this project and three at a time for handling purpose. These animals were maintained free of infection other than the treatments received in the study. All live animal-related protocols were approved by Committee on Animal Care and Use at Iowa State University.

Vaccine. M. paratuberculosis vaccine (Mycopar®) from Fort Dodge Animal Health was

used for vaccination of calves. Mycopar is a whole cell bacterin containing inactivated M. paratuberculosis bacteria suspended in oil. Six animals were given an injection of 0.25 ml

(half dose) M. paratuberculosis vaccine subcutaneously on the left side of the neck at Day 0.

Three control animals received equal volume of saline instead at Day 0.

Bacterial inoculum and infection.The M. paratuberculosis lab-adapted strain 19698

Middlebrook 7H9 broth supplemented with mycobactin J. Log-phase growing live M. paratuberculosis were resuspended in sterile saline for the use of in vivo infection. The

concentration of bacteria was determined as described previously (Chiodini and Buergelt, 1993) by measuring absorbance at 540nm and comparing the absorbance OD value against the standard curve. The viability of M. paratuberculosis inoculum used in these studies was

shown to be above 90% alive as checked via Fluorescein Diacetate (FDA) stain and flow cytometry analysis (Jayapal et al., 1991; Simutis et al., 2007). The purity of the inoculum was also checked. All nine animals were injected 5x108 CFU (colony forming units) of live M. paratuberculosis subcutaneously on the right side of the neck 60 days post vaccination, both

vaccinated and non-vaccinated groups.

Experimental design. Calves were either vaccinated or given saline (control) at the start of the study (Day 0) on the left side of the neck. 60 days post vaccination; all vaccinated and non-vaccinated animals were challenged subcutaneously on the right side of the neck with live M. paratuberculosis. Superficial cervical lymph nodes draining the M. paratuberculosis

inoculation site were removed aseptically 60 days post M. paratuberculosis inoculation.

Lymph nodes used for histology were fixed in 10% neutral buffered formalin. Lymph nodes used for cell isolation were transported back to laboratory in cold RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 0.5mM 2-mercaptoethanol, penicillin G (100 U/ml), streptomycin (100 µg/ml), amphotericin B (250 ng/ml), 2mM L-glutamine (complete medium).

On the day of excision, lymph nodes were processed to isolate total lymph node cells and to be used for further isolation of CD11c+ cells and lymphocytes (described below). Total lymph node cells were lysed and plated onto Middlebrook 7H10 agar for CFU (colony forming unit) count. Lymph node cells were also stained for selected cell type specific markers, including CD4, CD8, CD11c CD205 (DC), CD14 CD68 (macrophage), and B cell, to determine the cellular composition. Lymph nodes CD11c+ cells re-stimulated with PPD

(purified protein derivatives) were co-cultured with homogeneous lymph node lymphocytes for 7 days. Lymphocytes in the culture were then harvested to analyze the antigen-specific proliferation and intracellular cytokine production. Culture supernatant was collected to determine the cytokine release.

Peripheral blood was drawn one week before and on the day of lymph node removal. Peripheral blood cells were used to generate monocyte-derived dendritic cells (MD-DC) and to isolate lymphocytes used as responder cell donor in the proliferation assays.

Lymph node CD11c+ DC-like cells and lymphocytes isolation. Lymph nodes were minced to release the cells. Homogenization of the lymph nodes was also done when necessary. Above 90% cell viability can be achieved after the cell isolation in our hands. Total lymph node cells were then divided into groups for cellular composition analysis and for CD11c+ cells and lymphocytes separation.

Total lymph node cells were labeled with anti-bovine CD11c mAb (IgM, BAQ153A, VMRD), and followed by anti-IgM magnetic beads (Miltenyi Biotec). Labeled CD11c+ cells were positively selected using auto magnetic cell sorting (AutoMACS) separation column (Miltenyi Biotec). The CD11c+ cells isolated via this method were of above 90% purity as confirmed by flow cytometry. In order to recover lymph node lymphocytes, total lymph node cells were plated overnight in tissue culture flasks in complete medium to allow cells to adhere to the plastic surface. Non-adherent lymphocytes were washed off from the flasks with PBS and collected on the second day.

Peripheral blood monocyte-derived dendritic cell (MD-DC) generation and lymphocyte isolation. PBMC were isolated by density gradient centrifugation on Histopaque (1.083 g/ml, Sigma) and were collected from the interphase. Peripheral blood lymphocytes were separated based on failure to adhere to plastic culture flask surface.

MD-DC were generated following the protocol as described previously (Hope et al., 2003; Langelaar et al., 2005b; Lei and Hostetter, 2007; Werling et al., 1999). Briefly, PBMC

were labeled with mouse anti-bovine CD14 mAb (IgG1, MM61A, VMRD) and rat anti- mouse IgG1 magnetic microbeads (Miltenyi Biotec). CD14+ cells were positively selected using AutoMACS separation column and cultured in complete medium containing 10ng/ml recombinant human (rh) GM-CSF (PeproTech), 10ng/ml rhIL-4 (PeproTech) and 5ng/ml rhFlt3-ligand (R&D Systems) for 7 days. Half of the medium were replaced by fresh medium containing cytokines at Day 4.

Antibodies, reagents and flow cytometry. Single cell suspension of total lymph node cells was checked for cellular composition and the number of these different types of cells via flow cytometry (FACScan, Becton Dickinson). 106 lymph node cells were used for each marker staining.

Lymph node cells were incubated with relevant mAb: anti-bovine CD11c

(BAQ153A, VMRD), CD205 (CC98, Serotec), CD14 (M-M8, VMRD), B lymphocytes (B- B4, BAQ155A, VMRD), CD4 (CACT138A, VMRD), CD8-β (BAT82A, VMRD), CD25 (IL-2 receptor alpha, CACT116A, VMRD), MHC I (H58A, VMRD), MHC II (TH81A5, VMRD), and anti-human CD68 (EMB11, Dako), which had been diluted in FACS buffer (PBS containing 1% BSA and 0.05% sodium azide). Proper isotype controls were used at the same concentration as the antibody of interest in the study, including MOPC-31C (IgG1), MPC-11 (IgG2b), G155-178 (IgG2a), and G155-228 (IgM) (BD Pharmingen). Cells were then labeled with FITC (Fluorescein isothiocyanate) conjugated anti-IgG (Invitrogen), or PE (Phycoerythrin) conjugated anti-IgG (Invitrogen), or PE conjugated anti-IgM secondary antibodies (BD Pharmingen). Labeled cells were fixed in 2% paraformaldehyde and analyzed via flow cytometry. Gates were set based on the forward- and side-scatter profiles. Data were collected and analyzed using CellQuest (BD Bioscience) and FlowJo software (Tree Star, Inc.).

Histology of lymph nodes draining M. paratuberculosis inoculation sites. M. paratuberculosis inoculation site draining lymph nodes were fixed in 10% neutral buffered

formalin and sections of the tissue were stained with hematoxylin and eosin (H&E).

PPD-antigen specific T lymphocyte proliferation induced by M. paratuberculosis

challenge site draining lymph node CD11c+ _{cells. Isolated CD11c}+ _{cells from}

M. paratuberculosis challenge site draining lymph nodes (CLN) in vaccinated or non-vaccinated

animals were incubated in medium containing 10μg/ml PPD for overnight. PPD pulsed CLN CD11c+ cells were then added into 96-well round-bottom plate. Homogenous lymphocytes were separated from same challenge site lymph node. Lymphocytes were labeled with PKH67 green fluorescent cell linker (PKH67, Sigma) and added into the plate at a ratio of 1 CD11c+ cell to 10 lymphocytes. As negative and positive controls, labeled lymphocytes were cultured with medium alone or medium containing PPD or Concanavalin A (ConA, Sigma). Cells were kept in 37ºC, 5% CO2 incubator for 7 days.

At day 7, lymphocytes were collected and stained with either anti-bovine CD4 mAb (CACT138A) or anti-bovine CD8-β mAb (BAT82A). Proliferation of these T cells was analyzed by measuring the density of PKH67 fluorescence on the live CD4+ or CD8+ T cells via flow cytometry. Number of CD4+ or CD8+ T cells in the culture was determined by comparing with the known number of polystyrene latex microsphere (5μm, Invitrogen) added into the cell suspension. Data were collected and processed using CellQuest and Modfit LT software (Verity Software House).

Intracellular cytokine staining. PKH67 labeled CLN lymphocytes that had been co- cultured with PPD treated CD11c+ cells for 7 days were stained for intracellular cytokines production. Lymphocytes were fixed with 2% paraformaldehyde for 20 minutes and permeabilized with PermaWash buffer (PBS containing 0.1% saponin and 0.1% sodium azide) for 10 minutes. For IFN-γ staining, permeabilized lymphocytes were incubated with mouse anti-bovine IFN-γ mAb (7B6, IgG1, AbD Serotec) and followed by PE conjugated

anti-IgG secondary antibody (Invitrogen). For intracellular IL-4 staining, PE conjugated mouse anti-bovine IL-4 mAb (CC303, IgG2a, AbD Serotec) was used for the direct labeling. Proper isotype controls were included in the assay. Labeled lymphocytes were fixed again with 2% paraformaldehyde, washed, and analyzed via flow cytometry.

Enzyme-linked immunosorbent assay (ELISA). Supernatant of CLN lymphocyte-DC co-cultures was analyzed for secretion of cytokines IL-12 and IL-10 by ELISA. IL-12 and IL-10 ELISA were done following the protocols in the previous report (Lei and Hostetter, 2007). Briefly, mouse anti-bovine IL-12 mAb (CC301) 8μg/ml was used as capture antibody to coat plate. After blocking with 1% BSA (bovine serum albumin), test samples and controls were added. 0.5μg/ml biotinylated mouse anti-bovine IL-12 mAb (CC326) was used as detection antibody. For IL-10 ELISA, 5μg/ml mouse anti-bovine IL-10 mAb (CC318) and 2μg/ml biotinylated mouse anti-bovine IL-10 mAb (CC320) were used as capture and detection antibody respectively. All of these antibodies were obtained from AbD Serotec. Statistical analysis. Data are presented as mean value ± SEM (standard error mean) except where stated otherwise. Software used for statistical analysis and scientific graph generating include JMP 7 (SAS Institute Inc.) and GraphPad Prism 4.0 (GraphPad Software Inc.). Student’s t test and ANOVA (analysis of variance) test were used for the statistical analysis. Differences were considered significant if p < 0.05.

Results

Cell infiltration and cellular composition in the M. paratuberculosis challenge site draining lymph nodes (CLN) from vaccinated or non-vaccinated animals. 60 days post M. paratuberculosis inoculation, lymph nodes draining the M. paratuberculosis

challenge site (CLN) in both vaccinated and non-vaccinated animals were enlarged. Histologically, fewer germinal centers were present in the node cortex in CLN from non- vaccinated animals compared to the CLN from vaccinated animals (Figure 1). Total lymph node cells were lysed and plated on 7H10 agar plate. No CFU were recovered from the

lymph node draining M. paratuberculosis challenge site after one month culture, which was

independent of previous vaccination status (data not shown).

Given the differences identified by histological analysis, we conducted a flow

cytometric analysis of DC, macrophage, CD4 T cell, CD8 T cell, and B cell populations from the two groups following M. paratuberculosis challenge. Total lymph node cells were

labeled with selected cell type specific markers to determine the cellular composition inside the draining lymph node 60 days post inoculation. As shown in Figure 2A, CLN from both groups contained large numbers of B cells, CD4 T cells, and CD8 T cells. Although CD11c+ CD205+ cells and macrophages (CD14+ CD68+) were not the majority population in the lymph nodes, non-vaccinated animal CLN contained significantly fewer CD11c+ cells than vaccinated CLN (P < 0.05). B cells were also present at significantly lower numbers in non- vaccinated CLN, which correlates to the few germinal centers inside the lymph node (P < 0.05). No difference was seen in the numbers of macrophages, CD4 T cells, and CD8 T cells between two groups. Less than 10% CD4+ T cells expressed CD25 marker on the cell surface in both groups. Overall, CLN from vaccinated and non-vaccinated animals contained similar numbers of CD4+ CD25+ T cells (Figure 2B).

Phenotypic features of CD11c+ cells in the lymph nodes draining M.

paratuberculosis inoculation site (CLN). CD11c+ cells were assessed for selected

markers including CD14, CD205 (DEC205), MHC I and MHC II surface expression by flow cytometry (Figure 3). Lymph node CD11c+ cells expressed negative (below detection) CD14 on the cell surface, which suggested that these cells were not macrophages (CD14high). Lymph node CD11c+ cells expressed medium to high level CD205 marker independent of infection status (Figure 3), which belongs to the macrophage mannose receptor family of C- type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (Gliddon et al., 2004; Reiling et al., 2002). In vaccinated animals, CLN CD11c+ cells tended to have a higher level of CD205 surface expression compared to non-

vaccinated animals (Figure 3), also the numbers of CD205high CD11c+ cells were higher in vaccinated animal CLN (data not shown). As expected for lymph node DC-like cells, expression of MHC I and MHC II on lymph node CD11c+ cells was high (Figure 3). Vaccinated animal CLN CD11c+ cells expressed highest level of MHC I and MHC II on surface. Non-vaccinated animal CLN DC expressed similar levels of MHC I and MHC II as DC in saline control LN. The percentage of cells positive for these two markers was about the same between lymph nodes from vaccinated and non-vaccinated groups, while both were significantly higher than monocyte-derived dendritic cells (p < 0.05, data not shown).

Function of CD11c+ DC-like cells in the lymph node draining M. paratuberculosis inoculation site (CLN). In order to evaluate the function of CD11c+ cells in the CLN, the capacity of these cells to induce lymph node CD4 T (Figure 4) and CD8 T (Figure 5)